Prokaryotic organisms provided with both an inner and outer cell membrane such as gram-negative bacteria only rarely secrete proteins out of the cell into the surrounding medium. Such proteins which do not remain in the cytoplasm are usually exported across the cytoplasmic membrane into the periplasmic space but do not cross the outer cell membrane. However, more recently examples have been found of proteins which are truly secreted from gram-negative bacteria.
Thus, Lysobacter enzymogenes secretes an alkaline phosphatase (cf. S. Au et al., J. Bacteriol. 173(15), 1991, pp. 4551-4557) and an .alpha.-lytic protease. Attempts to express the .alpha.-lytic protease in E. coli have resulted in production of the enzyme within cells as well as in the extracellular medium (cf. J. L. Silen et al., J. Bacteriol. 171(3), 1989, pp. 1320-1325).
Likewise, Achromobacter lyticus produces an extracellular protease, the primary structure of which appears from S. Tsunasawa et al., J. Biol. Chem. 264(7), 1989, pp. 3832-3839. The gene encoding A. lyticus protease I was cloned in E. coli in which the enzyme was exported to the periplasm rather than secreted into the culture medium (cf. T. Ohara et al., J. Biol. Chem. 264(34), 1989, pp. 20625-20631).